DNA analytic and hybridization methods to specifically detect and identify small amounts of nucleic acids are now indispensable to molecular biology, the genetic engineering industry, and molecular medicine. Of these, procedures that amplify target nucleic acid sequences-such as the polymerase chain reaction (PCR).sup.6, ligase amplification, and transcription-based systems derived from PCR-have proven most effective.sup.2.
Difficulties in critical analytic steps following DNA amplification, however, have prevented the technology from being more widely applied in clinical laboratories and the biotechnology industry. Analysis of DNA products following amplification usually takes one of three forms: (1) colorimetric hybridization analysis, (2) cloning, (3) direct sequencing.sup.1-3. Each of these procedures has proved more difficult than initially anticipated, in part because of special properties of amplified DNA that differ from nonamplified DNA. Numerous investigators have documented these well-known difficulties in analyzing amplified DNA, resulting from the following:
1. Complementary amplified DNA strands are usually uneven (or "ragged") because some strands are usually incompletely extended, and extraneous nucleotides are often added to 3' ends. "Ragged" DNA ends cannot be ligated to blunt end vectors, and is inefficiently bound by modifying enzymes.sup.1-6. PA1 2. Independently of the "raggedness" of DNA ends, base pairing at extreme DNA ends is believed to be unstable (an effect referred to as "breathing" of DNA ends). This effect appears to lead to increased susceptibility to exonuclease, and further difficulties in binding modifying enzymes, including restriction endonucleases. PCR-amplified DNA is widely found to cut inefficiently by many restriction endonucleases-including NotI, XbaI, XhoI, and SmaI-preventing analysis and cloning by many strategies.sup.5. PA1 3. Incompletely used substrates and reaction by products of amplification procedures can interfere with subsequent analysis. Interfering reactants, include nucleotides and oligonucleotides; interfering byproducts include pyrophosphates. PA1 4. Extraneous DNA, including "primer dimer," is often coamplified with target, confusing subsequent analysis and competing with target in cloning and sequencing steps. For this reason, amplified DNA must be extensively purified prior to these analytic procedures. PA1 5. Amplified DNA strands tend to rapidly reanneal and exclude hybridized probes.sup.1-3,6. This effect has been noted to create special difficulties in the sequencing of double-stranded PCR products. Numerous strategies have been suggested for converting PCR fragments into single strands so that they can be more easily analyzed by hybridization, sequencing, and cloning, including asymmetric PCR and magnetic beads. However, none has proved entirely satisfactory. PA1 Other strategies require sequence additions, such as 40 nt GC clamps, RNA promoters, restriction endonucleases etc. Since the needs of individual experiments vary greatly, investigators often need several oligonucleotides of similar or identical 3' sequence targeted to particular genes under study, but have various different 5' modifications. The current invention aims in large part to simplify and unify many different protocols requiring different 5' additions to amplified DNA. Defined 6-12 base sequences are added to the 5' of each amplification primer. After resulting amplification, three defined 6-12 base sequences are used to attach universal zipper adapter primers, so that useful desired chemical groups and functional sequences can be easily added to amplified DNA. Thus, the invention is a unified, integrated system to create an "economy of primers," so that an individual amplification product can be used in many different capacities without resynthesizing amplification primers.
Many strategies to circumvent difficulties in analyzing amplified DNA require oligonucleotide primers that have 5' modifications, such as chemical groups including biotin, digoxigenin and fluorescein that can aid in detection and/or purification of amplified DNA.
The invention also simplifies detection and characterization of amplified DNA. Previously, colorimetric PCR hybridization assays have been investigated as an-alternative to gel electrophoresis and Southern blot analysis of PCR-amplified DNA. Such gels and Southern blots have proven too time-consuming and tedious for typical clinical laboratories.sup.8. Solid-phase colorimetric PCR assays capture denatured amplification products on probes bound to nylon membranes (as in "reverse dot blots".sup.6-7) or to microtiter plates.sup.2,3. However, the sensitivity of these assays suffers due to the tendency of the denatured PCR product strands to reassociate and exclude oligonucleotide probes, and stearic interference between the bound oligonucleotides and the solid support, which impedes hybridization to nucleic acids in solution.sup.5,6. In some cases, colorimetric detection is improved by creating single-strand PCR products through asymmetric PCR that can associate with bound probes without interference.sup.7,9. Unfortunately, asymmetric PCR is notoriously difficult to reproduce, and does not lend itself to automation. Reamplification of the PCR product using internal or "nested" primers may improve assay sensitivity, but is costly, and compounds DNA contamination problems in clinical laboratories with concomitant false positive results.
Numerous different cloning procedures for analyzing amplified DNA can be found in the art, but all remain inefficient, expensive, and tedious. Restriction endonucleases do not cut PCR produced DNA well, and untreated PCR products cannot be cloned into blunt-end vectors unless the ragged ends of PCR are first repaired. Blunt end ligation is inefficient under any circumstances..sup.4
"TA" cloning vectors exploit the 5' adenosine residues that are sometimes added to the 3' of PCR products to clone into a vector with thymidine residues (Invitrogen, San Diego, Calif.). Major problems with the use of these vectors includes instability of the terminal thymidine residue, inefficient transformation, and the limitation of using Taq polymerase to generate the terminal adenosine residues on the PCR product. Hybridization of single-base overhangs is inefficient, and these vectors do not work well in most laboratories.
Methods using T4 DNA polymerase to clone PCR products have recent been introduced into the art. These methods seek to introduce extended cohesive ends into PCR products complementary to mirror cohesive ends placed through recombinant methods into vectors. Stoker et al. performed PCR amplification with two primers which are homologous to the cohesive termini created by AccI and XmaI, respectively. The PCR products are treated with T4 DNA polymerase to remove 3' terminal sequences. After heat inactivation of the polymerase, the products are ligated to plasmid cut with AccI and XmaI.sup.10. Aslanidis et al. generated clonable PCR fragments with 5' ends containing an additional 12 nucleotide sequence which lacks dCMP.sup.11. After amplification, products were digested by T4 DNA polymerase in the presence of dGTP; the fragments thus have 5'-extending single-stranded tails of a defined sequence and length. In the same way, the plasmid vector was amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12 nucleotide tails complementary to the tails used for fragment amplification, permitting the creation of single-stranded ends with T4 DNA polymerase in the presence of dCTP.sup.11. This was similar to the method of Kuijper et al. ("prime" cloning) who introduced sequences into plasmids and lambda phage deficient in the nucleotide dTTP.sup.12. After digestion with restriction endonucleases and T4 DNA polymerase in the presence of dTTP, these vectors accept PCR fragments with mirror cohesive ends. Other investigators have used this technique with variable success.sup.13-15.
Each of the methods for cloning using T4 DNA polymerase suffers from severe difficulties. First, sequences to be made cohesive must be specially engineered into the vector either by PCR.sup.11 or via gene construction.sup.116. These methods are therefore unsuitable for use with most commonly used vectors. Second, PCR products must be extensively purified prior to cloning. The 3' exonuclease of T4 DNA polymerase is active only in the absence of 3 of 4 nucleotide species. Third, T4 DNA polymerase is found to be quite labile, and most commercially available lots of this enzyme are found to be unsuitable for this purpose. Users of these methods have documented the need to test multiple enzyme lots prior to identifying suitable T4 enzymes.
Kaling et al. replaced T4 DNA polymerase with exonuclease III as an enzyme for creating cohesive ends in PCR cloning.sup.5. However, this procedure requires kinased primers, and generates very limited cohesive ends (4 bases) corresponding to 5' protruding restriction endonuclease sites in plasmid. This method is therefore restricted to plasmids with appropriate restriction endonuclease sites. In addition, exonuclease III is often found to contain single-strand nuclease activity that can digest away cohesive protruding single-strand ends and lower cloning efficiency.
Cloning procedure using uracil DNA glycosylase (UDG).sup.17 are found in the art.sup.18-20. However, these procedures apply only to specially constructed vectors containing uracil (dUTP). Specially constructed PCR primers made with uracil phosphoramidite are also required, material that is expensive and unavailable to many laboratories.
Analysis of amplified DNA sequence can also be accomplished by direct sequencing without cloning to vectors. However, most laboratories have found sequencing of double-stranded PCR product to be inefficient and unsuitable for routine use. Several methods for converting PCR fragments into single strands, including the asymmetric PCR protocol of Gyllensten and Erlich.sup.9, the affinity strand separation method of Mitchell and Merrill.sup.21, and the magnetic strand separation method of Uhlen and coworkers.sup.22. However, the efficiency of asymmetric PCR is notoriously variable-from sample-to sample, and the other methods require expensive materials including biotinylated primers and streptavidin-coated beads.
Consequently, there remain several needs in the art for DNA analytic procedures to specifically detect, identify, and manipulate amplified DNA. In DNA diagnostics, the greatest need is a rapid, economical, highly sensitive colorimetric DNA hybridization test to specifically detect amplification products without running electrophoresis gels or performing Southern blot experiments. Preferably, such a test could be performed in a format familiar to clinical laboratories, such as a microtiter plate, with greater sensitivity than current tests to detect denatured DNA. In many cloning experiments, a need-exists for a convenient rapid procedure to flexibly recombine PCR-generated DNA into vector plasmids and bacteriophage with high efficiency, without the need to alter vector by addition of sequence, or alteration with restriction endonuclease. In other experiments, it is also important that vectors can be altered for rapid transfer of adapted DNA. The procedure needs to bypass the need for T4 DNA polymerase, an enzyme of variable efficiency and stability. PCR products should not require extensive purification. In sequencing, a need exists for a rapid method to convert amplified DNA partly or wholly into single strands without expensive or time-consuming protocols, and to extend the number of base sequences obtained per sequencing reaction. The present invention provides such methods as well as many related advantages.